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Dystrophic epidermolysis bullosa (DEB) is a genodermatose that impairs expression of collagen type VII in the dermal epidermal junction causing severe blistering of skin and chronical wounds. Conventional treatment is only palliative and gene therapy could be a potential treatment. Parameters regarding control of cell quality should be taken in account when designing an ex vivo gene therapy approach. As such, the objective was to quantify normal levels of COL7A1 synthesized and secreted in vitro by epidermal and dermal cells as well as to establish a method for clonal selection of fibroblasts. Human fibroblasts (HFB) and keratinocytes (HKT) were isolated and expanded from foreskin (1 and 2), scalp (3) and polydactyly (4). Multiple vials were frozen for each sample. Quantification of COL7A1 from cell homogenates and culture supernatant was done in duplicate using sandwich ELISA (O.D 450nm). Single cells were isolated by serial dilution in 96 well plates to test colony formation. The wells were maintained until a colony of 50 cells was formed and further expanded for quantification of population doubling (PDL/day). The test was done twice using different vials from the same sample (hfb2) for standardization of the technique. COL7A1 concentration (μg/cell) calculated from the standard curve and adjusted by number of cells at sample collection for supernatant and cell homogenate, were respectively: 2.7/1.04(HFB1);14.6/1.02(HFB2);14.1/1.21(HFB 3);28.48/2.86(HKT3);16.77/1.14(HFN4);2.75/1.22(HKT4). In the first clonal isolation, 31 single cells were obtained and 38.7% had the ability to form colonies of 50 cells and average PDL/day 0.44±0.09. In the second 36 single cells were isolated and 38% had the ability to form colonies and PDL/day 0.43±0.13. Colonies expanded during 36 to 47 days. Single cells that didn't form colonies had limited proliferation rate. Normal fibroblasts and keratinocytes secreted collagen VII in varying degrees. Both cell types could be targeted for gene correction in DEB and anatomical site should be taken in consideration. In literature, targeting holoclones of keratinocytes is considered critical for efficiency and positive outcome, but clonal selection of fibroblast for gene therapy is not as well described. The serial dilution was reproducible and could be used as a tool to select modified fibroblast capable of amplifying and maintaining long term correction or as preselection before editing. [ABSTRACT FROM AUTHOR]